Small-Molecule Immunosuppressive Drugs and Therapeutic Immunoglobulins Differentially Inhibit NK Cell Effector Functions in vitro

Small-molecule immunosuppressive drugs (ISD) prevent graft rejection mainly by inhibiting T lymphocytes. Therapeutic immunoglobulins (IVIg) are used for substitution, antibody-mediated rejection (AbMR) and HLA-sensitized recipients by targeting distinct cell types. Since the effect of ISD and IVIg on natural killer (NK) cells remains somewhat controversial in the current literature, the aim of this comparative study was to investigate healthy donor's human NK cell functions after exposure to ISD and IVIg, and to comprehensively review the current literature. NK cells were incubated overnight with IL2/IL12 and different doses and combinations of ISD and IVIg. Proliferation was evaluated by 3[H]-thymidine incorporation; phenotype, degranulation and interferon gamma (IFNγ) production by flow cytometry and ELISA; direct NK cytotoxicity by standard 51[Cr]-release and non-radioactive DELFIA assays using K562 as stimulator and target cells; porcine endothelial cells coated with human anti-pig antibodies were used as targets in antibody-dependent cellular cytotoxicity (ADCC) assays. We found that CD69, CD25, CD54, and NKG2D were downregulated by ISD. Proliferation was inhibited by methylprednisolone (MePRD), mycophenolic acid (MPA), and everolimus (EVE). MePRD and MPA reduced degranulation, MPA only of CD56bright NK cells. MePRD and IVIg inhibited direct cytotoxicity and ADCC. Combinations of ISD demonstrated cumulative inhibitory effects. IFNγ production was inhibited by MePRD and ISD combinations, but not by IVIg. In conclusion, IVIg, ISD and combinations thereof differentially inhibit NK cell functions. The most potent drug with an effect on all NK functions was MePRD. The fact that MePRD and IVIg significantly block NK cytotoxicity, especially ADCC, has major implications for AbMR as well as therapeutic strategies targeting cancer and immune cells with monoclonal antibodies

HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

BACKGROUND: Direct cellular contact with stimulated T cells is a potent mechanism that induces cytokine production in human monocytes in the absence of an infectious agent. This mechanism is likely to be relevant to T cell-mediated inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Microparticles (MP) generated by stimulated T cells (MPT) display similar monocyte activating ability to whole T cells, isolated T cell membranes, or solubilized T cell membranes. We previously demonstrated that high-density lipoproteins (HDL) inhibited T cell contact- and MPT-induced production of IL-1beta but not of its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra). METHODOLOGY/PRINCIPAL FINDINGS: Labeled MPT were used to assess their interaction with monocytes and T lymphocytes by flow cytometry. Similarly, interactions of labeled HDL with monocytes and MPT were assessed by flow cytometry. In parallel, the MPT-induction of IL-1beta and sIL-1Ra production in human monocytes and the effect of HDL were assessed in cell cultures. The results show that MPT, but not MP generated by activated endothelial cells, bond monocytes to trigger cytokine production. MPT did not bind T cells. The inhibition of IL-1beta production by HDL correlated with the inhibition of MPT binding to monocytes. HDL interacted with MPT rather than with monocytes suggesting that they bound the activating factor(s) of T cell surface. Furthermore, prototypical pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL3 and CCL4 displayed a pattern of production induced by MPT and inhibition by HDL similar to IL-1beta, whereas the production of CCL2, like that of sIL-1Ra, was not inhibited by HDL. CONCLUSIONS/SIGNIFICANCE: HDL inhibit both MPT binding to monocytes and the MPT-induced production of some but not all cytokines, shedding new light on the mechanism by which HDL display their anti-inflammatory functions

Interleukin-1β production in human monocytes/macrophages is differentially regulated by MEK1 upon sterile and infectious inflammatory conditions

Deregulation of the production of IL-1 and its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra), plays an important role in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Relevant to the latter conditions direct cellular contact with stimulated T cells potently triggers cytokine production in human monocytes. Identification of signal transduction pathways specific to pathogenic induction of cytokines may lead to new therapeutic approaches. Two different stimuli were compared to investigate the implication of MEK1 and MEK2 in the control of IL-1 and sIL-1Ra production by human monocytes: (i) soluble extracts of plasma membranes from stimulated T cells (CEsHUT), mimicking cellular contact with T cells, i.e., chronic/sterile inflammatory conditions; and (ii) LPS that is relevant to infectious inflammation. The ATP-noncompetitive MEK1/2 (U0126) and MEK1 (PD98059) specific inhibitors diminished the expression (protein and mRNA) of IL-1 in CEsHUT-activated monocytes. In contrast, only the concomitant inhibition of MEK1 and MEK2 inhibited IL-1 production in LPS-activated monocytes, whereas the inhibition of MEK1 only did not affect IL-1 production. In CEsHUT- and LPS-activated monocytes, MEK1 inhibition slightly affected sIL-1Ra production that was significantly inhibited by U0126. These results suggest that MEK1 and MEK2 are differentially involved in the regulation of the IL-1 system upon chronic/sterile and infectious inflammatory conditions. MEK1 which is dispensable to IL-1 production in LPS-activated monocytes represents a potential therapeutic target whose inhibition could participate in the restoration of IL-1/sIL-1Ra balance in chronic/sterile inflammation without affecting regular responses to pathogens

Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls

Blood extracellular vesicles (EVs) play essential roles in cell–cell communication and their molecular cargo is a promising source of disease biomarkers. However, proteomic characterization of plasma-derived EVs is challenged by the presence of highly abundant plasma proteins, which limits the detection of less abundant proteins, and by the low number of EVs in biological fluids. The aim of this study was to investigate if the removal of abundant plasma proteins prior to EV isolation could improve plasma-derived EV characterization by LC–MS/MS and expand the proteome coverage. Plasma depletion was performed using a single-use spin column and EVs were isolated from only 100 µL of non-depleted and depleted plasma by size exclusion chromatography. Afterwards, EVs were characterized by nanoparticle tracking analysis and mass spectrometry–based proteomics using a data-independent acquisition approach. Depleted plasma-derived EVs had higher particle concentrations and particle-to-protein ratios. Depletion did increase the protein coverage with a higher number of identifications in EVs from depleted plasma (474 proteins) than from non-depleted (386 proteins). However, EVs derived from non-depleted plasma carried a slightly higher number of common EV markers. Overall, our findings suggest that plasma depletion prior to EV isolation by size exclusion chromatography provides higher yield and protein coverage, but slightly lower identification of EV markers. This study also showed the possibility to characterize the proteome of EVs derived from small plasma volumes, encouraging the clinical feasibility of the discovery of EV biomarkers

A novel MEK2/PI3Kdelta pathway controls the expression of IL-1 receptor antagonist in IFN-beta-activated human monocytes

IFN-beta and sIL-1Ra play crucial roles in the regulation of innate immunity and inflammation. IFN-beta, which is widely used to improve the course of relapsing, remitting multiple sclerosis, induces the production of sIL-1Ra in human monocytes through mechanisms that remain largely unknown. In this study, we identified PI3Kdelta and MEK2 as key elements that control sIL-1Ra production in isolated human monocytes activated by IFN-beta. Blockade of MEK2, but not of MEK1, by inhibitors and siRNA prevented IFN-beta-induced PI3Kdelta recruitment to the membrane, Akt phosphorylation, and sIL-1Ra production, suggesting that MEK2 acted upstream of PI3Kdelta. Furthermore, ERK1/2, the only identified substrates of MEK1/2 to date, are dispensable for sIL-1Ra production in response to IFN-beta stimulation. Upon IFN-beta activation, MEK2 and PI3Kdelta are translocated to monocyte membranes. These data suggest that MEK1 and MEK2 display different, nonredundant functions in IFN-beta signaling. That neither MEK1 nor ERK1/2 play a part in this mechanism is also an unexpected finding that gives rise to a better understanding of the MAPK signaling network. Together, these findings demonstrate that IFN-beta triggers an atypical MEK2/PI3Kdelta signaling cascade to regulate sIL-1Ra expression in monocytes. The premise that MEK1 and MEK2 play a part in the induction of the proinflammatory cytokine, IL-1beta in human monocytes provides a rationale for an alternative, IFN-beta-mediated pathway to induce/enhance sIL-1Ra production and thus, to dampen inflammation

Differential regulation of cytokine production by PI3Kdelta in human monocytes upon acute and chronic inflammatory conditions

Deregulated production of cytokines, including IL-1beta, IL-6 and TNF plays an important role in chronic inflammation. Relevant to this condition, direct cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes/macrophages. We previously demonstrated that PI3Ks regulate differential production of IL-1beta and its specific inhibitor secreted IL-1 receptor antagonist (sIL-1Ra) by human monocytes. Here we show that in contrast with PI3Kalpha, beta and gamma, PI3Kdelta accounts for most of the PI3K-dependent signaling ruling the production of IL-1beta, IL-6, TNF and sIL-1Ra in monocytes activated by cellular contact with stimulated T cells (mimicked by CHAPS-solubilized membranes of stimulated T cells, CE sHUT) and lipopolysaccharides (LPS); the latter stimuli being relevant to chronic/sterile and acute/infectious inflammation, respectively. Interestingly, PI3Kdelta activity dampened the production of pro-inflammatory cytokines in LPS-activated monocytes, but induced it in CE sHUT-activated cells. In both CE sHUT- and LPS-activated monocytes PI3Kdelta regulated cytokine transcript expression through the phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK3beta). The blockade of GSK3beta displayed inverse effects to those of PI3Kdelta blockade. Thus, by displaying opposite functions in conditions mimicking chronic/sterile and acute/infectious inflammation, i.e., by repressing pro-inflammatory cytokine expression in LPS-activated monocytes but inducing such mediators in T cell contact-activated monocytes, PI3Kdelta represents a potential therapeutic target specific to chronic/sterile inflammatory conditions

Blockade of T cell contact-activation of human monocytes by high-density lipoproteins reveals a new pattern of cytokine and inflammatory genes.

BACKGROUND: Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact. METHODOLOGY/PRINCIPAL FINDINGS: To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNgamma, CCL2, CCL5, TNF, IL-1beta, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1beta, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E(2)-both involved in inflammatory response-were triggered by T cell contact and inhibited in the presence of HDL. CONCLUSIONS/SIGNIFICANCE: These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL

Glatiramer acetate triggers PI3Kdelta/Akt and MEK/ERK pathways to induce IL-1 receptor antagonist in human monocytes

Glatiramer acetate (GA), an immunomodulator used in multiple sclerosis (MS) therapy, induces the production of secreted IL-1 receptor antagonist (sIL-1Ra), a natural inhibitor of IL-1beta, in human monocytes, and in turn enhances sIL-1Ra circulating levels in MS patients. GA is a mixture of peptides with random Glu, Lys, Ala, and Tyr sequences of high polarity and hydrophilic nature that is unlikely to cross the blood-brain barrier. In contrast, sIL-1Ra crosses the blood-brain barrier and, in turn, may mediate GA anti-inflammatory activities within the CNS by counteracting IL-1beta activities. Here we identify intracellular signaling pathways induced by GA that control sIL-1Ra expression in human monocytes. By using kinase knockdown and specific inhibitors, we demonstrate that GA induces sIL-1Ra production via the activation of PI3Kdelta, Akt, MEK1/2, and ERK1/2, demonstrating that both PI3Kdelta/Akt and MEK/ERK pathways rule sIL-1Ra expression in human monocytes. The pathways act in parallel upstream glycogen synthase kinase-3alpha/beta (GSK3alpha/beta), the knockdown of which enhances sIL-1Ra production. Together, our findings demonstrate the existence of signal transduction triggered by GA, further highlighting the mechanisms of action of this drug in MS